Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use

Abstract

Provided herein are modified Fc proteins comprising non-natural amino acid residues at site-specific positions, conjugates of the modified Fc proteins for therapy or diagnosis, compositions comprising the modified Fc proteins and conjugates thereof, methods of their production and methods of their use. The modified Fc proteins and conjugates are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

1 . An Fc protein comprising a polypeptide chain having one or more non-natural amino acid residues at specific sites selected from the group consisting of optimally substitutable positions in the polypeptide chain, or a post-translationally modified variant thereof. 
     
     
         2 .- 18 . (canceled) 
     
     
         19 . A method of making an Fc protein conjugate, comprising:
 (a) contacting a nucleic acid template encoding one or more polypeptide chains of an Fc protein to a reaction mixture, wherein the reaction mixture translates the nucleic acid template, and wherein the Fc protein has one or more non-natural amino acids at specific sites selected from the group consisting of heavy chain residues H404, H241, H222, H221, H225, H227, H230, H231, H232, H236, H282, H289, H330, H335, H422, H272, H274, H332, H333, H341, H342, H386, H392, H442, and H443 according to the EU index of Kabat in the polypeptide chain, or a post-translationally modified variant or an aglycosylated variant thereof, and wherein each non-natural amino acid residue is independently selected from a group consisting of ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine; and   (b) conjugating the Fc protein to one or more payloads via one or more linkers.   
     
     
         20 . The method of  claim 19 , wherein each of the one or more non-natural amino acid residues at specific sites selected from the group consisting of heavy chain residues H404, H241, H222, H221, H225, H227, H230, H231, H232, H236, H282, H289, H330, H335, H422, H272, H274, H332, H333, H341, H342, H386, H392, H442, and H443 according to the EU index of Kabat,
 wherein each non-natural amino acid residue is according to the formula:   
       
         
           
           
               
               
           
         
         and wherein each L is independently a divalent linker, 
         and wherein each R is a residue of a reactive group linked to a payload. 
       
     
     
         21 . The method of  claim 20 , wherein each R is a residue of a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl. 
     
     
         22 . The method of  claim 20 , wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene and substituted heteroarylene. 
     
     
         23 . The method of  claim 19 , wherein the Fc protein is aglycosylated. 
     
     
         24 . The method of  claim 23 , wherein the Fc protein has a higher thermal stability (Tm1) compared to the corresponding wild-type Fc protein. 
     
     
         25 . The method of  claim 19 , wherein the Fc protein is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 
     
     
         26 . The method of  claim 19 , wherein each of the one or more non-natural amino acid residues comprises a residue of a reactive moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl. 
     
     
         27 . The method of  claim 19 , wherein each non-natural amino acid residue is according to the formula: 
       
         
           
           
               
               
           
         
       
       wherein each L is independently a phenylene, and wherein each R is independently a residue of a functional group linked to a payload, and wherein the functional group is selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl. 
     
     
         28 . The method of  claim 19 , wherein each of the one or more non-natural amino acid residues is selected from the group consisting of: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine. 
     
     
         29 . The method of  claim 19 , wherein the non-natural amino acid residue is p-azido-L-phenylalanine. 
     
     
         30 . The method of  claim 19 , wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine. 
     
     
         31 . The method of  claim 19 , wherein the non-natural amino acid residue is p-acetyl-L-phenylalanine. 
     
     
         32 . The method of  claim 19 , wherein the nucleic acid template has one or more stop codons, and wherein the stop codons encode sites of non-natural amino acid incorporation in the Fc protein. 
     
     
         33 . The method of  claim 19 , wherein the one or more payloads are selected from the group consisting of: anti-tubulin agents, auristatins, maytansinoids, platinols, rapamycins, steroids, and topoisomerase inhibitors. 
     
     
         34 . The method of  claim 19 , wherein the one or more payloads are topoisomerase inhibitors. 
     
     
         35 . The method of  claim 19 , wherein the reaction mixture comprises one or more orthogonal tRNAs aminoacylated with the one or more non-natural amino acids, and wherein each orthogonal tRNA base pairs with a codon in the nucleic acid template that is not normally associated with an amino acid. 
     
     
         36 . The method of  claim 35 , wherein the codon is a stop codon. 
     
     
         37 . The method of  claim 35 , wherein the codon is a 4 bases codon. 
     
     
         38 . The method of  claim 19 , wherein the reaction mixture comprises one or more tRNA synthetases that aminoacylate one or more orthogonal tRNAs with the one or more non-natural amino acids. 
     
     
         39 . The method of  claim 19 , further comprising contacting translation release factors to the reaction mixture in step (a). 
     
     
         40 . The method of  claim 19 , further comprising contacting protein chaperones to the reaction mixture in step (a). 
     
     
         41 . The method of  claim 19 , wherein the Fc protein conjugate is made in a batch, an extended batch, a semi-batch, a semi-continuous, a fed-batch or a continuous reaction.